The present invention relates to methods for detecting viral pathogens, particularly human herpes virus 6 (HHV6), using polymerase chain reaction (PCR) techniques.
Cytomegalovirus (CMV) shedding and disease are classically associated with immunosuppressive therapy following organ transplantation in two distinct settings. Primary disease, often with organ involvement usually occurs within the first 6 weeks. The less severe secondary disease, two to three months after transplantation may be caused by reactivation or reinfection with CMV. Biological anti-rejection therapy, however, including OKT3 and anti-thymocyte globulins (ATG), is now the major risk factor for CMV disease in the CMV seropositive patient.
Other herpesviruses, including human herpes virus 6 (HHV6), reactivate during periods of intense immunosuppression. Infection with HHV6 is usual in the first one to three years of life and a minority develop exanthem subitum during primary infection. HHV6 antibody levels tend to decrease in adults over 30 years of age and may reach undetectable levels. Two major subspecies of HHV6, variants A and B, have been distinguished on genetic, antigenic and biological characteristics. Reactivation of HHV6 (variant B) has been reported in bone marrow, renal and liver transplant patients and has been associated with hepatitis, severe interstitial pneumonitis and encephalitis. Serologic evidence has been reported for simultaneous reactivation of CMV and HHV6 after renal transplantation and there have been reports of dual infection with CMV and either HHV6 or HHV7 in transplant patients. Prospective studies of the role of HHV6 in febrile disease following renal transplantation and the potential interaction between CMV and HHV6 reactivation in causing disease are lacking.
Viral markers which can accurately predict CMV/HHV6 disease, the need for antiviral therapy, and likelihood of successful response in renal transplant recipients is an important clinical priority. Buffy coat cultures have been a more reliable predictor of CMV disease in renal transplantation than detection in urine. More recently, detection of CMV DNA in plasma or quantification in urine or buffy coat have been shown to be predictive of disease in liver, renal transplant or HIV infected patients. HHV6 DNA has been detected in sera from immunosuppressed patients with HIV infection or undergoing bone marrow transplantation. The present inventors have examined prospectively reactivation or infection with CMV and HHV6 detected as viral DNA by polymerase chain reaction (PCR) in serum and urine, to determine the relative contributions of the two viruses towards disease during renal transplantation and to consider whether active infection of both viruses together may predict either the frequency or severity of disease.
The present inventors have detected an association between viral infections and have developed new methods to detect and differentiate viral infections in organ transplant and immunocompromised patients.
In a first aspect, the present invention consists in an isolated nucleic acid molecule complementary to and specific for human herpes virus 6 (HHV6) DNA including a sequence selected from the group consisting of 5xe2x80x2CTTCTGTTTTAAGTCGTACAGGAGT (SEQ ID NO: 1), 5xe2x80x2ACAAGTTGCCATTTCGGGGAAGTAC (SEQ ID NO: 2), and functionally equivalent sequences.
In a preferred embodiment of the present invention, the molecule consists of the sequence 5xe2x80x2CTTCTGTTTTAAGTCGTACAGGAGT (SEQ ID NO: 1), 5xe2x80x2ACAAGTTGCCATTTCGGGGAAGTAC (SEQ ID NO: 2), or a functional equivalent of either one of these sequences.
A functionally equivalent sequence is defined as a sequence being different by one or more bases but still specific for and able to bind to the DNA of HHV6.
In a second aspect, the present invention consists in a method for amplifying HHV6 DNA which method involves the use of a pair of oligonucleotide primers comprising the sequences 5xe2x80x2CTTCTGTTTTAAGTCGTACAGGAGT (SEQ ID NO: 1)and 5xe2x80x2ACAAGTTGCCATTTCGGGGAAGTAC (SEQ ID NO: 2), or including functionally equivalent sequences. Preferably, the primers consist of the sequences 5xe2x80x2CTTCTGTTTTAAGTCGTACAGGAGT (SEQ ID NO: 1)and 5xe2x80x2ACAAGTTGCCATTTCGGGGAAGTAC (SEQ ID NO: 2), or functionally equivalent sequences.
In a third aspect, the present invention consists in a method of detecting HHV6 in a sample containing HHV6, the method comprising the steps of:
(a) optionally, amplifying viral DNA present in the sample by polymerase chain reaction techniques using outer primers complimentary to the viral DNA;
(b) adding to the sample, or to the sample having undergone optional amplification step (a), a pair of inner oligonucleotide primers complementary to and specific for HHV6 DNA, wherein the inner primers include the sequences 5xe2x80x2AAGCTTGCACAATGCCAAAAAACAG (SEQ ID NO: 3) and 5xe2x80x2CTCGAGTATGCCGAGACCCCTAATC (SEQ ID NO: 4), or functionally equivalent sequences;
(c) carrying out polymerase chain reaction techniques on the sample so as to amplifying the HHV6 DNA spanned by the inner primers present in the sample; and
(d) detecting the amplified HHV6 DNA.
In a preferred embodiment of the third aspect of the present invention, the method comprises the steps of:
(a) optionally, amplifying viral DNA present in the sample by polymerase chain reaction techniques by
(i) adding outer primers, complimentary to the viral DNA in the sample,
(ii) providing buffers, reagents, nucleotides and a thermostable DNA polymerase to the sample to form a reaction mixture,
(iii) heating the reaction mixture to a temperature such that double stranded viral DNA present denatures to form single stranded DNA molecules,
(iv) cooling the reaction mixture to a temperature such that the outer primers anneal to their respective complementary sequences on the denatured single stranded DNA molecules,
(v) heating the reaction mixture to a temperature such that the DNA polymerase extends the primers to form new double stranded DNA molecules spanning the region of DNA defined by the outer primers, and
(vi) repeating steps (iii), (iv) and (v) such that the number of copies of the region of DNA encoding the double stranded viral DNA is amplified;
(b) adding to the optionally amplified sample a pair of inner oligonucleotide primers complementary to and specific for HHV6 DNA, wherein the inner primers include the sequences 5xe2x80x2AAGCTTGCACAATGCCAAAAAACAG (SEQ ID NO: 3)and 5xe2x80x2CTCGAGTATGCCGAGACCCCTAATC (SEQ ID NO: 4), or functionally equivalent sequences;
(c) heating the reaction mixture to a temperature such that the optionally amplified double stranded viral DNA denatures to form single stranded DNA molecules;
(d) cooling the reaction mixture to a temperature such that the inner primers anneal to their respective complementary sequences on the denatured DNA;
(e) heating the reaction mixture to a temperature such that the DNA polymerase extends the primers to form new double stranded DNA molecules spanning the region of DNA defined by the inner primers;
(f) repeating steps (c), (d) and (e) such that the number of copies of the region of DNA is amplified; and
(g) detecting the amplified DNA.
The sample can be any sample including fixed or frozen tissue samples, and any biological fluid including blood, serum, urine, semen, sputum, saliva, cerebrospinal spinal fluid, cord blood and other excretions. Preferably, the sample is serum or urine. The same can be pre-treated to extract or concentrate the nucleic acid material (DNA) from the sample by standard methods known in the art. Outer primers comprising the sequences 5xe2x80x2CTTCTGTTTTAAGTCGTACAGGAGT (SEQ ID NO: 1) and 5xe2x80x2ACAAGTTGCCATTTCGGGGAAGTAC (SEQ ID NO: 2) have been found to be particularly suitable for the optional steps of amplifying viral DNA in the sample. More preferably, the outer primers consist of the sequences 5xe2x80x2CTTCTGTTTTAAGTCGTACAGGAGT (SEQ ID NO: 3)and 5xe2x80x2ACAAGTTGCCATTTCGGGGAAGTAC (SEQ ID NO: 2).
Preferably, the inner primers consist of the sequences 5xe2x80x2CTTCTGTTTTAAGTCGTACAGGAGT (SEQ ID NO: 3)and 5xe2x80x2ACAAGTTGCCATTTCGGGGAAGTAC (SEQ ID NO: 4). It will be appreciated that other outer and inner primers based on the preferred primers according to the present invention and having slight differences in their sequences may also be suitable.
The detection of the amplified DNA in step (g) can be by any means known to the art. Preferably, the DNA is separated by electrophoresis and the DNA detected by a detectably-labelled viral specific probe. One such probe suitable includes the sequence 5xe2x80x2AACTGTCTGACTGGCAAAAACTTTT (SEQ ID NO: 5).
The term xe2x80x9cpolymerase chain reactionxe2x80x9d or xe2x80x9cPCRxe2x80x9d when used herein generally refers to a procedure where minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in the literature (1,2). Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical in sequence or similar in sequence to opposite strands of the template to be amplified. The 5xe2x80x2 terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc (3,4).
As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of an established nucleic acid (DNA or RNA) as a primer, and utilises a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid (5,6).
The terms xe2x80x9cligation chain reactionxe2x80x9d or xe2x80x9cLCRxe2x80x9d or xe2x80x9cligation amplification reactionxe2x80x9d or xe2x80x9cLARxe2x80x9d when used herein generally refer to a procedure where minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified (7,8). Generally, sequence information from the region of interest needs to be available, such that oligonucleotide pairs can be designed that are complementary to adjacent sites on an appropriate nucleic acid template. The oligonucleotide pair is ligated together by the action of a ligase enzyme. The amount of ligated product may be increased by either linear or exponential amplification using sequential rounds of such template-dependent ligation. In the case of linear amplification, a single pair of oligonucleotides is ligated, the reaction is heated to dissociate the ligation product from its template, and similar additional rounds of ligation are performed. Exponential amplification utilises two pairs of oligonucleotides, one pair being complementary to one strand of a target sequence and the other pair being complementary to the second strand of the target sequence. In this case the products of ligation serve as mutually complementary templates for subsequent rounds of ligation, interspersed with heating to separate the ligated products from the template strands. A single base-pair mismatch between the annealed oligonucleotides and the template prevents ligation, thus allowing the distinction of single base-pair differences between DNA templates.
LAR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc (7,8).
As used herein, LAR is considered to be one, but not the only, example of a nucleic acid ligase reaction method for amplifying a nucleic acid test sample, comprising the use of an established nucleic acid (DNA or RNA) as a primer/probe, and utilises a nucleic acid ligase to amplify or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid (9,10).
As an example, PCR methods and conditions found to be particularly suitable for the third aspect of the present invention are set out below. Specimen DNA was amplified in a final volume of 100 xcexcl of 10 mM Tris (pH 8.3), 50 mM KCI, 2.0 mM MgCl2, (2.5 mM for CMV) 0.01% gelatin, 0.1% Tritonxc3x97100, 0.2 mM of dNTPs, 20 pmoles of each outer primer and 1.5 units of Taq DNA polymerase (Promega, Madison, USA). After an initial denaturation for 3 minutes at 94xc2x0 C., the DNA was subjected to 30 cycles of amplification with 1 minute at 94xc2x0 C., 40 seconds at 55xc2x0 C. (60xc2x0 C. for CMV) and 1.5 minutes at 72xc2x0 C. followed by a final extension at 72xc2x0 C. for 7 mins in thermal cycler. When nested PCR was performed for HHV6, 2 xcexcl of the first product was transferred to a fresh tube containing similar concentrations of the reagents, except that the inner primers were used. Conditions of cycling were similar, but with 20 cycles for the primary amplification followed by 30 cycles for the nested. The amplified products were analysed by electrophoresis on an 8% polyacrylamide gel and hybridisation with a radiolabelled probe was carried out.
In a fourth aspect, the present invention consists in a method of detecting or diagnosing infection with HHV6 in a subject, the method including carrying out the method according to the third aspect of the present invention on a sample from the subject to detect the presence of HHV6 in the sample.
The present inventors have found that the method is particularly suitable for detecting the presence of HHV6 in organ transplant recipients and donors. It will be appreciated, however, that the method would be applicable for other uses where the detection or diagnosis of HHV6 is. required.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
In order that the present invention may be more clearly understood, preferred forms will be described in the following examples with reference to the accompanying drawings.